Within the human mouth, bacterial cell-cell communication and complex multispecies interactions regulate microbial gene expression, affecting the ecological balance of oral biofilms and human health. Many bacteria communicate using small signaling molecules, a process termed quorum sensing (QS). The basic QS model states that bacteria 'count' their cell numbers using signaling molecules, and modulate group behavior based upon population density. The concentration of these quorum signals within the human mouth is likely affected by multiple environmental factors, such as population size and flow of saliva or crevicular fluid across dental plaque, however it is not yet fully understood which factors regulate QS. This proposal aims to characterize the parameters that affect QS, and examine interactions in highly dense, small populations of three species of oral bacteria: Streptococcus gordonii, Aggregatibacter actinomycetemcomitans (Aa), and Streptococcus mutans. S. gordonii is a commensal and opportunistic pathogen, and colonizes adjacent to both Aa, the etiologic agent of localized aggressive periodontitis, and S. mutans, the primary causative agent of dental caries in humans. Current microbiology studies almost exclusively examine microbial communication and interactions on scales much larger than normally occur in the human mouth. We propose a change in the approach for probing microbial interactions using a novel methodology we developed to study smaller population sizes (d104 cells). With this technology, a single bacterium can be captured within a picoliter-sized trap made of cross-linked protein. Within these porous bacterial lobster traps, bacteria grow rapidly to a desired cell number enclosed in a three-dimensional user-defined geometry. The goal of this proposal is to determine whether phenotypes of large populations are relevant in small populations, and to determine how spatial distribution of species affects polymicrobial interactions. We plan to examine two previously characterized polymicrobial interactions in small communities within the traps: S. gordonii H2O2 production mediating Aa resistance to host innate immunity; and S. gordonii protease production inhibiting bacteriocin expression in S. mutans. We will probe for these interactions in picoliter-sized communities using GFP transcriptional reporters strains, and fluorescent live/dead stains. The goals of this project are in line with the mission of the National Institute of Dental and Craniofacial Research: the proposal aims to improve the understanding of communication and multispecies interactions of oral pathogens, and furthermore will advance the field of oral microbiology by introducing a novel, useful technique for studying population sizes relevant to infections and disease in the human mouth.